It has been recognized that crops grown in some soils are naturally resistant to certain fungal pathogens. Furthermore, soils that are conducive to the development of these diseases can be rendered suppressive or resistant to the pathogen by the addition of small quantities of soil from a suppressive field (Scher and Baker (1980) Phytopathology 70: 412-417). Conversely, suppressive soils can be made conducive to fungal disease susceptibility by autoclaving, indicating that the factors responsible for disease control are biological. Subsequent research has demonstrated that root colonizing bacteria are responsible for this phenomenon, which is known as biological disease control (Cook and Baker (1983), The Nature and Practice of Biological Control of Plant Pathogens; Amer. Phytopathol. Soc., St. Paul, Minn.).
In many cases, the most efficient strains of biological disease controlling bacteria are fluorescent pseudomonads (Weller et al. (1983) Phytopathology, 73: 463-469). These bacteria have also been shown to promote plant growth in the absence of a specific fungal pathogen by the suppression of detrimental rhizosphere microflora present in most soils (Kloepper et al. (1981) Phytopathology 71: 1020-1024). Important plant pathogens that have been effectively controlled by seed inoculation with these bacteria include Gaemannomyces graminis, the causative agent of take-all in wheat (Cook et al. (1976) Soil Biol. Biochem 8: 269-273) and Pythium and Rhizoctonia, pathogens that cause damping off of cotton (Howell et al. (1979) Phytopathology 69: 480-482). Rhizoctonia is a particularly problematic plant pathogen for several reasons. First, it is capable of infecting a wide range of crop plants, and second, there are no commercially available chemical fungicides that are effective in controlling the fungus.
Many biological disease controlling Pseudomonas strains produce antibiotics that inhibit the growth of fungal pathogens (Howell et al. (1979) Phytopathology 69:480-482; Howell et al. (1980) Phytopathology 70: 712-715). These antibiotics have been implicated in the control of fungal pathogens in the rhizosphere. For example, Howell et al. (Phytopathology 69: 480-482; 1979) disclose a strain of Pseudomonas fluorescens that produces an antibiotic substance antagonistic to Rhizoctonia solani. In addition, other strains of Pseudomonas fluorescens having enhanced biocontrol activity against plant pathogenic fungi such as Rhizoctonia and Pythium are disclosed in U.S. Pat. Nos. 5,348,742 and 5,496,547, both of which are hereby incorporated by reference in their entireties. Several other past studies have focused on the effects of mutations that result in the inability of the disease control bacterium to synthesize these antibiotics (Kloepper et al. (1981) Phytopathology 71: 1020-1024; Howell et al. (1983) Can. J. Microbiol. 29: 321-324). In these cases, the ability of the organism to control the pathogen is reduced, but not eliminated.
A particularly effective antibiotic against fungal pathogens is pyrrolnitrin, which is biosynthesized from tryptophan (Chang et al. J. Antibiot. 34: 555-566). Pyrrolnitrin is a phenylpyrrole derivative with strong antibiotic activity that has been shown to inhibit a broad range of fungi (Homma et al., Soil Biol. Biochem. 21: 723-728 (1989); Nishida et al., J. Antibiot., ser. A, 18: 211-219 (1965)). Pyrrolnitrin was originally isolated from Pseudomonas pyrrocinia (Arima et al, J. Antibiot., ser. A, 18: 201-204 (1965)), but has since been isolated from Myxococcus species, Burkholdaria species, and several other Pseudomonas species such as PS. FLUORESCENS (Gerth et al. J. Antibiot. 35: 1101-1103 (1982); J. N. Roitman, N. E. Mahoney and W. J. Janisiewicz, Applied Microbiology and Biotechnology 34:381-386 (1990)). The compound has been reported to inhibit fungal respiratory electron transport (Tripathi & Gottlieb, J. Bacteriol. 100: 310-318 (1969)) and uncouple oxidative phosphorylation (Lambowitz & Slayman, J. Bacteriol. 112: 1020-1022 (1972)). It has also been proposed that pyrrolnitrin causes generalized lipoprotein membrane damage (Nose & Arima, J. Antibiot., ser A, 22: 135-143 (1969); Carlone & Scannerini, Mycopahtologia et Mycologia Applicata 53: 111-123 (1974)). U.S. Pat. No. 5,639,949 and U.S. Pat. No. 5,817,502, both of which are hereby incorporated by reference in their entireties, describe the cloning and characterization of the pyrrolnitrin biosynthetic genes from Ps. fluorescens and Ps. pyrrocinia.
An important factor in biological control is the ability of a biocontrol organism to compete in a given environment (Baker et al. (1982) Biological Control of Plant Pathogens, American Phytopathological Society, St. Paul, Minn., pages 61-106). Thus, it is desirable to obtain strains of biocontrol agents that effectively control the growth of fungal pathogens such as Rhizoctonia and Pythium and that are also able to aggressively compete with indigenous bacteria and microflora existing in the rhizosphere of the plant.